The flower industry strives to develop new and different varieties of flowers. An effective way to create such novel varieties is the manipulation of flower color where classical breeding techniques have been used to produce a wide range of colors for most of the commercial varieties. This approach has been limited, however, by the constraints of a particular species' gene pool and for this reason it is rare for a single species to have the full spectrum of colored varieties.
Flower color is predominantly due to two types of pigments: flavonoids and carotenoids. Flavonoids mainly contribute a wide range of color from yellow to red to blue, whereas carotenoids mainly contribute color tones such as orange or yellow. The flavonoids which are a major contribution to flower color are a class of compounds called anthocyanins. The chromophoric group of anthocyanins is anthocyanidins, and as major anthocyanidins, there are known pelargonidin, cyanidin and delphinidin. Plants are known to have a wide variety of anthocyanins, and the diversity thereof is one of the causes of the diversity of flower colors. Structures of hundreds of anthocyanins have already been determined, and the hydroxyl group at the 3 position of most anthocyanins has been modified with sugars (Harbone, in The Flavonoids: 565, 1986).
The biosynthetic pathway for anthocyanins is common among flowering plants up to the biosynthesis of the 3-glucosides (Holton et al., Plant Cell 7: 1071, 1995), and subsequently they undergo various modifications such as glycosylation, acylation and methylation in species- and variety-specific manners. Such differences in modification patterns in varieties are one of the reason for diversities in anthocyanins, i.e. diversities in flower colors. Generally the more aromatic acyl groups modify anthocyanins, the more stabilized and bluer anthocyanins become (Harbone, in the Flavonoids: 565, 1986; Norio Saito, TANPAKUSITU KAKUSAN KOUSO (Proteins, Nucleic Acids, Enzymes) 47: 202, 2002). Furthermore, flower color may be affected by the formation of metal complex of anthocyanins, the copigment effect by flavonoid compounds such as flavonol and flavone, and pH of vacuoles in which anthocyanins are localized (Forkmann, Plant Breeding 106: 1, 1991).
Biosynthesis of flavonoids including anthocyanidin has been extensively studied. All the genes for enzymes involved in anthocyanin biosynthesis have been cloned, and genes for the transcription factors therefor have also been obtained. Therefore, the artificially modification of the expression of these genes can alter the structure and the amount of flavonoids accumulated in flowers, and thereby can change flower color. There are some reports on the modification of anthocyanin structures and flower color by a molecular biological technique and gene transformation into plants (Forkmann G. & Martens S. (2001), Curr. Opin. Biotechnology, 12: 155-160; Tanaka Y. & Mason J. (2003), In: Singh R P & Jaiwal P K (ed.) Plant genetic engineering, pp. 361-385, SCI tech publishing, Houston).
One possible method for making flower color blue is to increase the number of hydroxyl groups of B ring of anthocyanin. An enzyme that catalyzes a reaction of hydroxylating the 3′ position of anthocyanin (flavonoid 3′-hydroxylase: F3′H) and an enzyme that catalyzes a reaction of hydroxylating the 3′ and the 5′ position of anthocyanin (flavonoid 3′,5′-hydroxylase: F3′5′H) are important in altering flower color. In general, pelargonidin (one hydroxyl group in B ring) is contained in orange- to red-colored flowers, cyanidin (two hydroxyl groups in B ring) is contained in red- to magenta-colored flowers, and delphinidin (three hydroxyl groups in B ring) is contained in purple- to blue-colored flowers. In most cases, plant species that do not have purple- to blue-colored varieties often lack the ability to produce delphinidin, and are represented by roses, chrysanthemums and carnations.
For these plants, the creation of purple- to blue-colored varieties by biotechnology has long attracted attention. In fact, by expressing the F3′5′H gene essential for the production of delphinidin, carnations whose flower color is blue purple were produced (Tanaka Y. & Mason J. (2003), In: Singh R P & Jaiwal P K (ed.) Plant genetic engineering, pp. 361-385, SCI tech publishing, Houston), and it became possible to produce delphinidin in flower petals, but the flower color has not been fully blue yet. Thus, in order to make flower color sheer blue, the introduction of the F3′5′H gene alone is not sufficient, and further contrivances may be required.
Actually anthocyanins contained in blue flowers are often modified with aromatic acyl groups via sugars (Honda & Saito, Heterocycles 56: 633 (2002)). Thus, one possible method of making flower color blue is to modify anthocyanins with aromatic acyl groups such as caffeoyl groups, coumaroyl groups and sinapoyl groups (Tanaka Y. & Mason J. (2003), In: Singh R P & Jaiwal P K (ed.) Plant genetic engineering, pp. 361-385, SCI tech publishing, Houston).
Generally, anthocyanin is slightly reddened by glycosylation, and the addition of aromatic acyl groups via sugars makes the color of anthocyanin blue (Forkmann, Plant Breeding 106: 1, 1991). Also, anthocyanin is a compound unstable in neutral solutions, and the stability is enhanced by modification with sugars or acyl groups (Forkmann, Plant Breeding 106: 1, 1991). An experiment using anthocyanins from morning glories (Pharbitis nil) revealed that acylated anthocyanins to which an aromatic acyl group such as, for example, coumaric or caffeic acid was bound showed a hypsochromic shift (Dangle et al., Phytochemistry 34: 1119, 1993).
As for anthocyanins acylated with aromatic acyl groups, many isolation examples from nature have been reported including awobanin (Goto and Kondo, Angew. Chem. Int. Ed. Engl. 30: 17, 1991) derived from Commelina communis (Honda & Saito, Heterocycles 56: 633 (2002)). For example, anthocyanins from blue flowers have multiple aromatic acyl groups as represented by cinerarin (derived from cineraria), gentiodelphin (derived from Gentiana triflora), heavenly blue anthocyanin (derived from Pharbitis nil), ternatin (derived from Clitoria ternatea) and lobelinin (derived from Lobelia).
Cinerarin (Goto et al., Tetrahedron 25: 6021, 1984) derived from cineraria (Senecio cruentus) has one aliphatic acyl group and three aromatic acyl groups, and these aromatic acyl groups are reported to contribute to the stabilization of pigments in neutral aqueous solutions (Goto et al., Tetrahedron 25: 6021, 1984). Gentiodelphin (DEL 3G-5CafG-3′ CafG) which is a major pigment of Gentiana triflora petals has a delphinidin 3-glycoside as the basic backbone, and two side chains comprising one glucose molecule and one caffeic acid molecule on the hydroxyl groups at the 5 position and the 3′ position. It is reported that the side chains at the 5 and 3′ position comprised of sugar-acyl group contributed to a sandwich-type of intra-molecular stacking, resulting in the stabilization of pigments in aqueous solutions (Yoshida et al., Tetrahedron 48: 4313, 1992). Furthermore, it has been confirmed that among the two side chains of sugar-acyl group, the glucosylacyl group at the 3′ position rather than the 5 position contributes more strongly to the stabilization and blueing of pigments (Yoshida et al., Phytochemistry 54: 85, 2000).
The aromatic acyl transfer reaction was first demonstrated in Silene (Kamsteeg et al., Biochem. Physiol. Pflanzen 175: 403, 1980), a plant of the family Caryophyllaceae, in 1980, and a similar aromatic acyl transferase activity was also found in the solubilized enzyme fraction of Matthiola as well (Teusch et al., Phytochemistry 26: 991, 1986). Subsequently, an anthocyanin 5-aromatic acyltransferase (hereinafter 5AT) that transfers aromatic acyl groups such as caffeic acid and coumaric acid to sugars at the 5 position of anthocyanins was isolated from Gentiana triflora (Fujiwara et al., Eur. J. Biochem. 249, 45, 1997), and based on the internal amino acid sequences of the purified enzyme, cDNA that codes for 5AT of Gentiana triflora was isolated (Fujiwara et al., Plant J., 16, 421, 1998).
Based on this gene, a homolog was isolated from Torenia (WO 2005/017147), and furthermore based on the amino acid sequence conserved between these enzymes, a Perilla cDNA coding for the enzyme (3AT) that transfers aromatic acyl groups to the sugar at the 3 position of anthocyanins was isolated (Yonekura-Sakibara et al., Plant Cell Physiol 41: 495, 2000). Using the Perilla 3AT gene, the 3AT gene was cloned from lavender of the same family Labiatae (WO 1996/25500).
An enzyme gene that transfers an acyl group to anthocyanidin-3-rutinoside has been obtained from petunia (National Publication of Translated Version (Kohyo) No. 2003-528603). When the Perilla 3AT gene or the torenia 5AT gene was introduced into roses, anthocyanin in which aromatic acyl groups were added to the 3 position or the 5 position was formed in petals, but it failed to significantly alter flower color blue, and the maximum absorption spectra just shifted to the long wavelength by about 1-2 nm.
The reason for this, as reported by Yoshida et al. (Yoshida et al., Tetrahedron 48: 4313, 1992), it was thought that acylation of A ring or C ring such as the 3 or 5 position is not fully effective, and that acylation at the 3′ position is necessary for blueing and stabilization of an anthocyanin, and more preferably acylation at multiple positions including the 3′ position is necessary. Since there is in fact anthocyanins containing an aromatic acyl group attached to a sugar at the 3′ position, the presence of an enzyme (3′ AT) that catalyzes a reaction of transferring an aromatic acyl group to a sugar at the 3′ position may be postulated. However, there is no report on a measurement for 3′ AT reaction and no 3′ AT enzyme or a gene encoding for a 3′ AT has been isolated so far.
All acyltransferases reported so far act on the 3 position or the 5 position of anthocyanin, and the site specificity of the reaction has been reported to be high (Fujiwara et al., Plant J., 16, 421, 1998; Yonekura-Sakibara et al., Plant Cell Physiol 41: 495, 2000). Therefore, the acylation at the 3′ position using a known aromatic acyltransferase was thought to be impossible. There have been no report for an aromatic acyltransferase that have an activity of transferring aromatic acyl groups to multiple positions of anthocyanins. Thus, with the level of conventional technology, it was impossible, for example, to create a recombinant plant and transfer aromatic acyl groups to sugars at the 3′ position or multiple positions including the 3′ position of anthocyanin. That is, it was impossible to add aromatic acyl groups to sugars at the 3′ position or multiple positions including the 3′ position of an anthocyanin in order to make a bluer and more stable anthocyanin, and to make bluer and more stable flower color.    Patent document 1: WO 1996/25500    Patent document 2: WO 2005/017147    Patent document 3: National Publication of Translated Version (Kohyo) No. 2003-528603    Non-patent document 1: Harbone, in The Flavonoids: 565, 1986    Non-patent document 2: Holton et al., Plant Cell 7: 1071, 1995    Non-patent document 3: Harbone, in The Flavonoids: 565, 1986    Non-patent document 4: Norio Saito, TANPAKUSITU KAKUSAN KOUSO (Proteins, Nucleic Acids, Enzymes) 47: 202, 2002    Non-patent document 5: Forkmann, Plant Breeding 106: 1, 1991    Non-patent document 6: Forkmann G. & Martens S. (2001), Curr. Opin. Biotechnology, 12: 155-160    Non-patent document 7: Tanaka Y. & Mason J. (2003), In: Singh R P & Jaiwal P K (ed.) Plant genetic engineering, pp. 361-385, SCI tech publishing, Houston    Non-patent document 8: Honda & Saito, Heterocycles 56: 633 (2002)    Non-patent document 9: Forkmann, Plant Breeding 106: 1, 1991    Non-patent document 10: Dangle et al., Phytochemistry 34: 1119, 1993    Non-patent document 11: Goto et al., Tetrahedron 25: 6021, 1984    Non-patent document 12: Yoshida et al., Tetrahedron 48: 4313, 1992    Non-patent document 13: Yoshida et al., Phytochemistry 54: 85, 2000    Non-patent document 14: Goto and Kondo, Angew. Chem. Int. Ed. Engl. 30: 17, 1991    Non-patent document 15: Kamsteeg et al., Biochem. Physiol. Pflanzen 175: 403, 1980    Non-patent document 16: Teusch et al., Phytochemistry 26: 991, 1986    Non-patent document 17: Fujiwara et al., Eur. J. Biochem. 249, 45, 1997    Non-patent document 18: Fujiwara et al., Plant J., 16, 421, 1998    Non-patent document 19: Yonekura-Sakakibara et al., Plant Cell Physiol 41: 495, 2000